Major changes in the kinetic mechanism of AMP inhibition and AMP cooperativity attend the mutation of Arg49 in fructose-1,6-bisphosphatase

The significance of subunit interface residues Arg49 and Lys50 in the function of porcine liver fructose-1,6-bisphosphatase was explored by site-directed mutagenesis, initial rate kinetics, and circular dichroism spectroscopy. The Lys50 --> Met mutant had kinetic properties similar to the wild-type enzyme but was more thermostable. Mutants Arg49 --> Leu, Arg49 --> Asp, Arg49 --> Cys were less thermostable than the wild-type enzyme yet exhibited wild-type values for kcat and Km. The Ki for the competitive inhibitor fructose 2,6-bisphosphate increased 3- and 5-fold in Arg49 --> Leu and Arg49 --> Asp, respectively. The Ka for Mg2+ increased 4-8-fold for the Arg49 mutants, with no alteration in the cooperativity of Mg2+ binding. Position 49 mutants had 4-10-fold lower AMP affinity. Most significantly, the mechanism of AMP inhibition with respect to fructose 1,6-bisphosphate changed from noncompetitive (wild-type enzyme) to competitive (Arg49 --> Leu and Arg49 --> Asp mutants) and to uncompetitive (Arg49 --> Cys mutant). In addition, AMP cooperativity was absent in the Arg49 mutants. The R and T-state circular dichroism spectra of the position 49 mutants were identical and superimposable on only the R-state spectrum of the wild-type enzyme. Changes from noncompetitive to competitive inhibition by AMP can be accommodated within the framework of a steady-state Random Bi Bi mechanism. The appearance of uncompetitive inhibition, however, suggests that a more complex mechanism may be necessary to account for the kinetic properties of the enzyme.     

Identification of a phosphate regulatory site and a low affinity binding site for glucose 6-phosphate in the N-terminal half of human brain hexokinase

Crystal structures of human hexokinase I reveal identical binding sites for phosphate and the 6-phosphoryl group of glucose 6-phosphate in proximity to Gly87, Ser88, Thr232, and Ser415, a binding site for the pyranose moiety of glucose 6-phosphate in proximity to Asp84, Asp413, and Ser449, and a single salt link involving Arg801 between the N- and C-terminal halves. Purified wild-type and mutant enzymes (Asp84 --> Ala, Gly87 --> Tyr, Ser88 --> Ala, Thr232 --> Ala, Asp413 --> Ala, Ser415 --> Ala, Ser449 --> Ala, and Arg801 --> Ala) were studied by kinetics and circular dichroism spectroscopy. All eight mutant hexokinases have kcat and Km values for substrates similar to those of wild-type hexokinase I. Inhibition of wild-type enzyme by 1,5-anhydroglucitol 6-phosphate is consistent with a high affinity binding site (Ki = 50 microM) and a second, low affinity binding site (Kii = 0.7 mM). The mutations of Asp84, Gly87, and Thr232 listed above eliminate inhibition because of the low affinity site, but none of the eight mutations influence Ki of the high affinity site. Relief of 1,5-anhydroglucitol 6-phosphate inhibition by phosphate for Asp84 --> Ala, Ser88 --> Ala, Ser415 --> Ala, Ser449 --> Ala and Arg801 --> Ala mutant enzymes is substantially less than that of wild-type hexokinase and completely absent in the Gly87 --> Tyr and Thr232 --> Ala mutants. The results support several conclusions. (i) The phosphate regulatory site is at the N-terminal domain as identified in crystal structures. (ii) The glucose 6-phosphate binding site at the N-terminal domain is a low affinity site and not the high affinity site associated with potent product inhibition. (iii) Arg801 participates in the regulatory mechanism of hexokinase I.     

Function, structure and evolution of fructose-1,6-bisphosphatase

The hydrolysis of fructose-1,6-bisphosphate to fructose-6-phosphate is a key reaction of carbohydrate metabolism. The enzyme that catalyzes this reaction, fructose-1,6-bisphosphatase, appears to be present in all forms of living organisms. Regulation of the enzyme activity, however, occurs by a variety of distinct mechanisms. These include AMP inhibition (most sources), cyclic AMP-dependent phosphorylation (yeast), and light-dependent activation (chloroplast). In this short review, we have analyzed the function of several fructose-1,6-bisphosphatases and we have made a comparison of partial amino acid sequences obtained from the enzymes of the yeast Saccharomyces cerevisiae, Escherichia coli, and spinach chloroplasts with the known entire amino acid sequence of a mammalian gluconeogenic fructose-1,6-bisphosphatase. These results demonstrate a very high degree of sequence conservation, suggesting a common evolutionary origin for all fructose-1,6-bisphosphatases.     

Selection and characterization of beta-lactam-beta-lactamase inactivator-resistant mutants following PCR mutagenesis of the TEM-1 beta-lactamase gene

Mechanism-based inactivators of beta-lactamases are used to overcome the resistance of clinical pathogens to beta-lactam antibiotics. This strategy can itself be overcome by mutations of the beta-lactamase that compromise the effectiveness of their inactivation. We used PCR mutagenesis of the TEM-1 beta-lactamase gene and sequenced the genes of 20 mutants that grew in the presence of ampicillin-clavulanate. Eleven different mutant genes from these strains contained from 1 to 10 mutations. Each had a replacement of one of the four residues, Met69, Ser130, Arg244, and Asn276, whose substitutions by themselves had been shown to result in inhibitor resistance. None of the mutant enzymes with multiple amino acid substitutions generated in this study conferred higher levels of resistance to ampicillin alone or ampicillin with beta-lactamase inactivators (clavulanate, sulbactam, or tazobactam) than the levels of resistance conferred by the corresponding single-mutant enzymes. Of the four enzymes with just a single mutation (Ser130Gly, Arg244Cys, Arg244Ser, or Asn276Asp), the Asn276Asp beta-lactamase conferred a wild-type level of ampicillin resistance and the highest levels of resistance to ampicillin in the presence of inhibitors. Site-directed random mutagenesis of the Ser130 codon yielded no other mutant with replacement of Ser130 besides Ser130Gly that produced ampicillin-clavulanate resistance. Thus, despite PCR mutagenesis we found no new mutant TEM beta-lactamase that conferred a level of resistance to ampicillin plus inactivators greater than that produced by the single-mutation enzymes that have already been reported in clinical isolates. Although this is reassuring, one must caution that other combinations of multiple mutations might still produce unexpected resistance.     

The diaphragm and respiratory muscles

A rapid procedure for the purification of the redox-regulated chloroplast fructose-1,6-bisphosphatase [EC 3.1.3.11] from spinach leaf extract to homogeneity is described. No thiol-reducing agents were present during the purification and the enzyme is > 99% in the oxidized form. A rapid procedure to reduce and activate the Fru-1,6-P2ase by dithiothreitol in the absence of thioredoxin is described. Reduction activates the enzyme up to several hundred-fold when assayed at pH 8.0 with 2 mM Mg2+. The activity of the purified oxidized enzyme is unusually sensitive to changes in Mg2+ and H+ concentration. Tenfold changes in Mg2+ or H+ concentration lead to > 100-fold increases in activity. The recoveries of fructose-1,6-bisphosphatase activity as determined by the activity of the oxidized enzyme at pH 8.0/20 mM Mg2+; pH 9.0/2 mM Mg2+; pH 8/2 mM Mg2+ plus 0.1 mM Hg(II) or of the reduced enzyme at pH 8.0/2 mM Mg2+ are similar (approximately 40%) indicating that the major proportion of these activities in a leaf extract is catalyzed by the same enzyme. Moreover, antibodies raised against the purified enzyme inhibit all of the above activities in crude leaf extracts. The kinetic properties of the purified enzyme suggest that the oxidized Mg(2+)-dependent enzyme can play no significant role in photosynthetic carbon assimilation. A survey of some kinetic properties of Fru-1,6-P2ase activity in extracts of various photosynthetic organisms reveals that all 11 species examined possess a redox- and pH/Mg(2+)-stimulated Fru-1,6-P2ase, whereas Fru-1,6-P2ase in extracts of Taxus baccata (a gymnosperm), Chlorella vulgaris (a green alga), and the cyanobacterium Nostoc muscorum were not activated by Hg(II). The heat stability that proved useful in the purification of the spinach enzyme was conserved in both angiosperms and gymnosperms. The oxidized enzyme (which normally has no thiol groups accessible to 5,5'-dithio-bis[2-nitrobenzoic acid]) but not the reduced enzyme can be stimulated many hundred-fold by addition of extraordinarily low concentrations of Hg(II) to a complete assay mixture. With the aid of EDTA as a Hg(II) buffer, half-maximal stimulation was achieved at 2 x 10(-16) M free Hg(II). Methylmercury also stimulates the enzyme many hundred-fold at very low concentrations. The concentration for half-maximal stimulation by methylmercury was determined with a cyanide buffer to be approximately 10(-16) M. This, together with the high affinity of the enzyme for Hg(II), suggests that Hg(II) stimulates the enzyme by binding to an enzyme thiol group that be comes exposed in the catalytically active enzyme, thereby stabilizing the oxidized enzyme in an active conformation. By contrast, in the absence of Fru-1,6-P2 and either Mg2+ or Ca2+, Hg(II) (even at 2 x 10(-16) M) rapidly inactivates the oxidized Fru-1,6-P2ase. This inactivation is similar to the inactivation of Fru-1,6-P2ase that occurred at high pH (> 9) and which is also prevented by Fru-1,6-P2 and either Mg2+ or Ca2+. Although the Hg(II)- and high pH-inactivated oxidized enzyme has no activity, both forms of the enzyme can be activated by reduction. The usefulness of buffers to maintain low, defined Hg(II) and organic mercurial concentrations is discussed.     

Partial activation of muscle phosphorylase by replacement of serine 14 with acidic residues at the site of regulatory phosphorylation

Phosphorylation of glycogen phosphorylase at residue Ser14 triggers a conformational transition that activates the enzyme. The N-terminus of the protein, in response to phosphorylation, folds into a 310 helix and moves from its location near a cluster of acidic residues on the protein surface to a site at the dimer interface where a pair of arginine residues form charged hydrogen bonds with the phosphoserine. Site-directed mutagenesis was used to replace Ser14 with Asp and Glu residues, analogs of the phosphoserine, that might be expected to participate in ionic interactions with the arginine side chains at the dimer interface. Kinetic analysis of the mutants indicates that substitution of an acidic residue in place of Ser14 at the site of regulatory phosphorylation partially activates the enzyme. The S14D mutant shows a 1.6-fold increase in Vmax, a 10-fold decrease in the apparent dissociation constant for AMP, and a 3-fold decrease in the S0.5 for glucose 1-phosphate. The S14E mutant behaves similarly, showing a 2.2-fold increase in Vmax, a 6-fold decrease in the apparent dissociation constant for AMP, and a 2-fold decrease in the S0.5 for glucose 1-phosphate. The ability of the mutations to enhance binding of AMP and glucose 1-phosphate and to raise catalytic activity suggests that the introduction of a carboxylate side chain at position 14 promotes docking of the N-terminus at the subunit interface and concomitant stabilization of the activated conformation of the enzyme. Like the native enzyme, both mutants show significant activity only in the presence of the activator, AMP. Full activation, analogous to that provided by covalent phosphorylation of the enzyme, likely is not achieved because of differences in the charge and the geometry of ionic interactions at the phosphorylation site.     

Role of a dynamic loop in cation activation and allosteric regulation of recombinant porcine fructose-1,6-bisphosphatase

A disordered loop (loop 52-72, residues 52-72) in crystal structures of fructose-1,6-bisphosphatase (FBPase) has been implicated in regulatory and catalytic phenomena by studies in directed mutation. A crystal structure of FBPase in a complex with three zinc cations and the products fructose 6-phosphate (F6P) and phosphate (Pi) reveals loop 52-72 for the first time in a well-defined conformation with strong electron density. Loop 52-57 interacts primarily with the active site of its own subunit. Asp68 of the loop hydrogen bonds with Arg276 and a zinc cation located at the putative potassium activation site. Leu56 and Tyr57 of the loop pack against hydrophobic residues from two separate subunits of FBPase. A mechanism of allosteric regulation of catalysis is presented, in which AMP, by binding to its allosteric pocket, displaces loop 52-72 from the active site. Furthermore, the current structure suggests that both the alpha- and beta-anomers of F6P can be substrates in the reverse reaction catalyzed by FBPase. Mechanisms of catalysis are proposed for the reverse reaction in which Asp121 serves as a catalytic base for the alpha-anomer and Glu280 serves as a catalytic base for the beta-anomer.     

Analysis of the stabilization of hen lysozyme by helix macrodipole and charged side chain interaction

In the N-terminal region of the alpha-helix of the c-type lysozymes, two Asx residues exist at the 18th and 27th positions. Hen lysozyme has Asp18/Asn27 (18D/27N), and we prepared three mutant lysozymes, Asn18/Asn27 (18N/27N), Asn18/Asp27 (18N/27D), and Asp18/Asp27 (18D/27D). The stability of the wild-type (18D/27N) lysozyme supported the existence of a hydrogen bond between the side chain of Asp18 and the amide group at the N1 position in the alpha-helix, while the stability of the 18N/27D lysozyme supported the presence of the capping box between the Ser24 (N-cap) and Asp27 residues. Although electrostatic repulsion was observed between Asp18 and Asp27 residues in 18D/27D lysozyme, the dissociation of each residue contributed to stabilizing the B-helix in 18D/27D lysozyme through hydrogen bonding and charge-helix macrodipole interaction. This is the first evidence that two neighboring negative charges at the N-terminus of the helix both increased the stability of the protein.     

Catalytic domain of phosphoinositide-specific phospholipase C (PLC). Mutational analysis of residues within the active site and hydrophobic ridge of plcdelta1

Structural studies of phospholipase C delta1 (PLCdelta1) in complexes with the inositol-lipid headgroup and calcium identified residues within the catalytic domain that could be involved in substrate recognition, calcium binding, and catalysis. In addition, the structure of the PLCdelta1 catalytic domain revealed a cluster of hydrophobic residues at the rim of the active site opening (hydrophobic ridge). To assess a role of each of these residues, we have expressed, purified, and characterized enzymes with the point mutations of putative active site residues (His311, Asn312, Glu341, Asp343, His356, Glu390, Lys438, Lys440, Ser522, Arg549, and Tyr551) and residues from the hydrophobic ridge (Leu320, Phe360, and Trp555). The replacements of most active site residues by alanine resulted in a great reduction (1,000-200,000-fold) of PLC activity analyzed in an inositol lipid/sodium cholate mixed micelle assay. Measurements of the enzyme activity toward phosphatidylinositol, phosphatidylinositol 4-monophosphate, and phosphatidylinositol 4, 5-bis-phosphate (PIP2) identified Ser522, Lys438, and Arg549 as important for preferential hydrolysis of polyphosphoinositides, whereas replacement of Lys440 selectively affected only hydrolysis of PIP2. When PLC activity was analyzed at different calcium concentrations, substitutions of Asn312, Glu390, Glu341, and Asp343 resulted in a shift toward higher calcium concentrations required for PIP2 hydrolysis, suggesting that all these residues contribute toward Ca2+ binding. Mutational analysis also confirmed the importance of His311 ( approximately 20,000-fold reduction) and His356 ( approximately 6,000-fold reduction) for the catalysis. Mutations within the hydrophobic ridge, which had little effect on PIP2 hydrolysis in the mixed-micelles, resulted in an enzyme that was less dependent on the surface pressure when analyzed in a monolayer. This systematic mutational analysis provides further insights into the structural basis for the substrate specificity, requirement for Ca2+ ion, catalysis, and surface pressure/activity dependence, with general implications for eukaryotic phosphoinositide-specific PLCs.     

A case of cytophagic histiocytic panniculitis: successful treatment of recurrent attacks with steroid pulse therapy and oral cyclosporin A

Basic fibroblast growth factor (bFGF) is implicated in the pathogenesis of several vascular and connective diseases. A key step in the discovery of bFGF receptor antagonists to mitigate these actions is to define the functional epitope required for receptor binding of the growth factor. In previous studies, we identified Glu96 as an essential residue in this epitope using site-directed mutagenesis. Here we examined the role of solvent accessible neighboring residues of Glu96 of bFGF on receptor binding affinity. Wild-type bFGF and its muteins were cloned and expressed in Escherichia coli and evaluated for FGF receptor binding affinity. Replacement of Asn104 of bFGF by alanine reduced receptor binding affinity over 400-fold compared with wild-type bFGF. We next explored the effect of neighboring residues of Asn104 on receptor binding affinity-Muteins in which Arg97, Leu98, Glu99, Asn101, Asn102, Thr105 and Pro141 were individually replaced by alanine exhibited receptor binding similar to wild-type bFGF. By contrast, substitution of Tyr103 or Leu140 by alanine reduced receptor binding affinity about 400- and 150-fold, respectively, in accord with a previous report. We conclude that at least six solvent-accessible residues in bFGF are crucial for high-affinity receptor binding, as evidenced by at least a 10-fold diminution in the affinity of the corresponding alanine muteins. The polar residues Glu96 and Asn104 appear to form an area important for facilitating the initial contact between ligand and receptor, whereas Tyr24, Tyr103, Leu140 and Met142 form a hydrophobic patch that may stabilize the complex. The detailed structure of this functional epitope can be employed in the discovery and design of bFGF antagonists using computational methods.     

Looking for residues involved in the muscle acylphosphatase catalytic mechanism and structural stabilization: role of Asn41, Thr42, and Thr46

Asn41, Thr42, and Thr46 are invariant residues in both muscle and erythrocyte acylphosphatases isolated so far. Horse muscle acylphosphatase solution structure suggests their close spatial relationship to Arg23, the main substrate binding site. The catalytic and structural role of such residues, as well as their influence on muscle acylphosphatase stability, was investigated by preparing several gene mutants (Thr42Ala, Thr46Ala, Asn41Ala, Asn41Ser, and Asn41Gln) by oligonucleotide-directed mutagenesis. The mutated genes were cloned and expressed in Escherichia coli, and the mutant enzymes were purified by affinity chromatography and investigated as compared to the wild-type enzyme. The specific activity and substrate affinity of Thr42 and Thr46 mutants were not significantly affected. On the contrary, Asn41 mutants showed a residual negligible activity (about 0.05-0.15% as compared to wild-type enzyme), though maintaining an unchanged binding capability of both substrate and inorganic phosphate, an enzyme competitive inhibitor. According to the 1H nuclear magnetic resonance spectroscopy and circular dichroism results, all mutants elicited well-constrained native-like secondary and tertiary structures. Thermodynamic parameters, as calculated from circular dichroism data, demonstrated a significantly decreased stability of the Thr42 mutant under increasing temperatures and urea concentrations. The reported results strongly support a direct participation of Asn41 to the enzyme catalytic mechanism, indicating that Asn41 mutants may well represent a useful tool for the investigation of the enzyme physiological function by the negative dominant approach.     

Identification of a genetic mutation in a family with fructose-1,6- bisphosphatase deficiency

Fructose-1,6-bisphosphatase deficiency is an inheritable disorder of gluconeogenesis. Sequence analysis of the cDNA of the fructose-1,6-bisphosphatase mRNA isolated from monocytes from a girl with this disease and her consanguineous parents revealed that the patient and her parents were a homozygote and heterozygotes for an insertion of one G residue at G957GGGG961, respectively. This mutation resulted in translation of a truncated enzyme protein, and the mutant protein showed no fructose-1,6- bisphosphatase activity in an overexpression experiment in Escherichia coli. However, this mutation is located in a region of the amino acid sequence which is not well conserved among mammals. A mutagenized clone was prepared from the normal clone. The extents of substitutions and deletions of the amino acid sequence were predicted to be less in the mutagenized protein than in the mutant protein. This mutagenized clone also expressed no fructose-1,6-bisphosphatase activity, although both of two normal clones from control monocytes and a control liver sample expressed an apparently normal level of fructose-1,6-bisphosphatase activity. Thus, this mutation is concluded to be responsible for fructose-1,6-bisphosphatase deficiency in this patient.     

Regulation of fructose-1,6-bisphosphatase activity in primary cultured astrocytes

In the gluconeogenic pathway, fructose-1,6-bisphosphatase (EC 3. 1. 3. 11) is the last key-enzyme before the synthesis of glucose-6-phosphate. The extreme diversity of cells present in the whole brain does not facilitate in vivo study of this enzyme and makes it difficult to understand the regulatory mechanisms of the related carbohydrate metabolism. It is for instance difficult to grasp the actual effect of ions like potassium, magnesium and manganese on the metabolic process just as it is difficult to grasp the effect of different pH values and the influence of glycogenic compounds such as methionine sulfoximine. The present investigation attempts to study the expression and regulation of fructose-1,6-bisphosphatase in cultured astrocytes. Cerebral cortex of new-born rats was dissociated into single cells that were then plated. The cultured cells were flat and roughly polygonal and were positively immunostained by anti-glial fibrillary acidic protein antibodies. Cultured astrocytes are able to display the activity of fructose-1,6-bisphosphatase. This activity was much higher than that in brain tissue in vivo. Fructose-1,6-bisphosphatase in cultured astrocytes did not require magnesium ions for its activity. The initial velocity observed when the activity was measured in standard conditions was largely increased when the enzyme was incubated with Mn2+. This increase was however followed by a decrease in absorbance resulting in the induction, by the manganese ions, of a singular kinetics in the enzyme activity. Potassium ions also stimulated fructose-1,6-bisphosphatase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conserved active site aspartates and domain-domain interactions in regulatory properties of the sugar kinase superfamily

The structures of the sugar kinase/heat shock 70/actin superfamily of enzymes show that the active site is located in a deep cleft between two domains whose relative movement defines a domain closure conformational change thought to be involved in the catalytic and regulatory properties of members of the superfamily. To investigate the role of the domain closure in the regulatory behavior, site-directed mutagenesis is used to alter specific domain-domain interactions in Escherichia coli glycerol kinase (EC 2.7.1.30; ATP:glycerol 3-phosphotransferase), a member of this superfamily. Two active site aspartate residues are conserved throughout the superfamily, one (Asp245 in glycerol kinase) which is proposed to act as a general base during catalysis and one (Asp10 in glycerol kinase) which interacts with the Mg(II) ion of the bound Mg(II)-nucleotide complex. Each of these residues participates in domain-domain interactions that are mediated by the bound substrates. The enzymes containing the substitutions Asp245 to Asn (D245N) or Asp10 to Asn (D10N) were purified by affinity chromatography, and the effects of the substitutions on the catalytic properties and regulation by the allosteric effectors, fructose 1,6-bisphosphate (FBP), and the glucose-specific phosphocarrier protein, IIIGlc (also known as IIAGlc), were determined. Each of the residues participates in catalysis; kcat/Katp is decreased 300-fold by the D245N substitution and 100-fold by the D10N substitution. Affinity labeling with the glycerol analog 1,3-dichloroacetone shows that the level of activity seen for the D245N mutant enzyme is not due to deamidation of the substituted asparagine. Each of the substitutions has little effect on regulation by FBP and the apparent affinity for IIIGlc, and the D245N substitution does not affect the extent of inhibition by IIIGlc. However, the D10N substitution decreases the maximum extent of inhibition by IIIGlc from 100 to 60%, thus changing the action of IIIGlc to that of a partial inhibitor. The different sensitivities of the extents of FBP and IIIGlc inhibition to perturbation of a domain-domain interaction mediated by Asp10 suggest that the relations of the actions of these allosteric effectors to the domain closure conformational change are different.     

The active sites of fructose 6-phosphate,2-kinase: fructose-2, 6-bisphosphatase from rat testis. Roles of Asp-128, Thr-52, Thr-130, Asn-73, and Tyr-197

To investigate the role in catalysis and/or substrate binding of the Walker motif residues of rat testis fructose 6-phosphate, 2-kinase:fructose-2,6-bisphosphatase (Fru 6-P,2-kinase:Fru-2,6-Pase), we have constructed and characterized mutant enzymes of Asp-128, Thr-52, Asn-73, Thr-130, and Tyr-197. Replacement of Asp-128 by Ala, Asn, and Ser resulted in a small decrease in Vmax and a significant increase in Km values for both substrates. These mutants exhibited similar pH activity profiles as that of the wild type enzyme. Mutation of Thr-52 to Ala resulted in an enzyme with an infinitely high Km for both substrates and an 800-fold decreased Vmax. Substitution of Asn-73 with Ala or Asp caused a 100- and 600-fold increase, respectively in KFru 6-P with only a small increase in KATP and small changes in Vmax. Mutation of Thr-130 caused small changes in the kinetic properties. Replacement of Tyr-197 with Ser resulted in an enzyme with severely decreased binding of Fru 6-P with 3-fold decreased Vmax. A fluorescent analog of ATP, 2'(3')-O-(N-methylanthraniloyl)ATP (mant-ATP) served as a substrate with Km = 0.64 microM, and Vmax = 25 milliunits/mg and was a competitive inhibitor with respect to ATP. When mant-ATP bound to the enzyme, fluorescence intensity at 440 nm increased. mant-ATP binding of the wild type and the mutant enzymes were compared using the fluorometric method. The Kd values of the T52A and D128N enzymes were infinitely high and could not be measured, while those of the other mutant enzymes increased slightly. These results provide evidence that those amino acids are involved in substrate binding, and they are consistent with the crystallographic data. The results also suggest that Asp-128 does not serve as a nucleophile in catalysis, and since there are no other potential nucleophiles in the active site, we hypothesize that the Fru 6-P,2-kinase reaction is mediated via a transition state stabilization mechanism.     

Importance of intramembrane carboxylic acids for occlusion of K+ ions at equilibrium in renal Na,K-ATPase

Site-directed mutagenesis and assay of Rb+ and Tl+ occlusion in recombinant Na,K-ATPase from yeast were combined to establish structure-function relationships of amino acid side chains involved in high-affinity occlusion of K+ in the E2[2K] form. The wild-type yeast enzyme was capable of occluding 2 Rb+ or Tl+ ions/ouabain binding site or alpha 1 beta 1 unit with high apparent affinity (Kd(Tl+) = 7 +/- 2 microM), like the purified Na,K-ATPase from pig kidney. Mutations of Glu327(Gln,Asp), Asp804(Asn, Glu), Asp808(Asn, Glu) and Glu779(Asp) abolished high-affinity occlusion of Rb+ or Tl+ ions. The substitution of Glu779 for Gln reduced the occlusion capacity to 1 Tl+ ion/alpha 1 beta 1-unit with a 3-fold decrease of the apparent affinity for the ion (Kd(Tl+) = 24 +/- 8 microM). These effects on occlusion were closely correlated to effects of the mutations on K0.5(K+) for K+ displacement of ATP binding. Each of the four carboxylate residues Glu327, Glu779, and Asp804 or Asp808 in transmembrane segments 4, 5, and 6 is therefore essential for high-affinity occlusion of K+ in the E2[2K] form. These residues either may engage directly in cation coordination or they may be important for formation or stability of the occlusion cavity.     

A comparison of gene organization in the zwf region of the genomes of the cyanobacteria Synechococcus sp. PCC 7942 and Anabaena sp. PCC 7120

The region of the genome encoding the glucose-6-phosphate dehydrogenase gene zwf was analysed in a unicellular cyanobacterium, Synechococcus sp. PCC 7942, and a filamentous, heterocystous cyanobacterium, Anabaena sp. PCC 7120. Comparison of cyanobacterial zwf sequences revealed the presence of two absolutely conserved cysteine residues which may be implicated in the light/dark control of enzyme activity. The presence in both strains of a gene fbp, encoding fructose-1,6-bisphosphatase, upstream from zwf strongly suggests that the oxidative pentose phosphate pathway in these organisms may function to completely oxidize glucose 6-phosphate to CO2. The amino acid sequence of fructose-1,6-bisphosphatase does not support the idea of its light activation by a thiol/disulfide exchange mechanism. In the case of Anabaena sp. PCC 7120, the tal gene, encoding transaldolase, lies between zwf and fbp.     

An ADP-ribosylation-factor(ARF)-like protein involved in regulated secretion

In higher plants, light enhances the activity of chloroplast fructose-1,6-bisphosphatase via a cascade of thiol/disulfide exchanges. We have examined the structural and functional role of seven conserved cysteine residues in the rapeseed (Brassica napus) enzyme by site-directed mutagenesis. After lysis of Escherichia coli cells, C53S and C191S variants partitioned mainly in the insoluble fraction whereas C96S, C157S, C174S, C179S, and C307S mutants were soluble. Homogeneous preparations of the latter hydrolyzed fructose 1,6-bisphosphate at similar rates in the presence of 10 mM Mg2+ but only C157S, C174S and C179S mutants were both efficient catalysts at 1 mM Mg2+ and nearly insensitive to dithiothreitol. These results demonstrate the contribution of Cys53 and Cys191 to the stability of the enzyme and the participation of Cys157, Cys174 and Cys179 in the reductive process responsive of the light-dependent regulation. Given that mutations at Cys96 and Cys307 neither destabilize the enzyme nor affect the reductive modulation, their function remains unknown.     

Role of electrostatic interactions on the affinity of thioredoxin for target proteins. Recognition of chloroplast fructose-1, 6-bisphosphatase by mutant Escherichia coli thioredoxins

Chloroplast thioredoxin-f functions efficiently in the light-dependent activation of chloroplast fructose-1, 6-bisphosphatase by reducing a specific disulfide bond located at the negatively charged domain of the enzyme. Around the nucleophile cysteine of the active site (-W-C-G-P-C-), chloroplast thioredoxin-f shows lower density of negative charges than the inefficient modulator Escherichia coli thioredoxin. To examine the contribution of long range electrostatic interactions to the thiol/disulfide exchange between protein-disulfide oxidoreductases and target proteins, we constructed three variants of E. coli thioredoxin in which an acidic (Glu-30) and a neutral residue (Leu-94) were replaced by lysines. After purification to homogeneity, the reduction of the unique disulfide bond by NADPH via NADP-thioredoxin reductase proceeded at similar rates for all variants. However, the conversion of cysteine residues back to cystine depended on the target protein. Insulin and difluoresceinthiocarbamyl-insulin oxidized the sulfhydryl groups of E30K and E30K/L94K mutants more effectively than those of wild type and L94K counterparts. Moreover, the affinity of E30K, L94K, and E30K/L94K E. coli thioredoxin for chloroplast fructose-1,6-bisphosphatase (A0.5 = 9, 7, and 3 microM, respectively) increased with the number of positive charges, and was higher than wild type thioredoxin (A0.5 = 33 microM), though still lower than that of thioredoxin-f (A0.5 = 0.9 microM). We also demonstrated that shielding of electrostatic interactions with high salt concentrations not only brings the A0.5 for all bacterial variants to a limiting value of approximately 9 microM but also increases the A0.5 of chloroplast thioredoxin-f. While negatively charged chloroplast fructose-1,6-bisphosphatase (pI = 4.9) readily interacted with mutant thioredoxins, the reduction rate of rapeseed napin (pI = 11.2) diminished with the number of novel lysine residues. These findings suggest that the electrostatic interactions between thioredoxin and (some of) its target proteins controls the formation of the binary noncovalent complex needed for the subsequent thiol/disulfide exchange.     

Mutation of serine-46 to aspartate in the histidine-containing protein of Escherichia coli mimics the inactivation by phosphorylation of serine-46 in HPrs from gram-positive bacteria

Histidine-containing protein (HPr) is a phosphocarrier protein of the bacterial phosphoenolpyruvate:sugar phosphotransferase system. HPr is phosphorylated at the active site residue, His15, by phosphoenolpyruvate-dependent enzyme I in the first enzyme reaction in the process of phosphoryl transfer to sugar. In many Gram-positive bacterial species HPr may also be phosphorylated at Ser46 by an ATP-dependent protein kinase but not in the Gram-negative Escherichia coli and Salmonella typhimurium. One effect of the phosphorylation at Ser46 is to make HPr a poor acceptor for phosphorylation at His15. In Bacillus subtilis HPr, the mutation Ser46Asp mimics the effects of phosphorylation. A series of mutations were made at Ser46 in E. coli HPr: Ala, Arg, Asn, Asp, Glu, and Gly. The two acidic replacements mimic the effects of phosphorylation of Ser46 in HPrs from Gram-positive bacteria. In particular, when mutated to Asp46, the His 15 phosphoacceptor activity (enzyme I Km/Kcat) decreases by about 2000-fold (enzyme I Km, 4 mM HPr; Kcat, approximately 30%). The alanine and glycine mutations had near-wild-type properties, and the asparagine and arginine mutations yielded small changes to the Km values. The crystallographic tertiary structure of Ser46Asp HPr has been determined at 1.5 A resolution, and several changes have been observed which appear to be the effect of the mutation. There is a tightening of helix B, which is demonstrated by a consistent shortening of hydrogen bond lengths throughout the helix as compared to the wild-type structure. There is a repositioning of the Gly54 residue to adopt a 3(10) helical pattern which is not present in the wild-type HPr. In addition, the higher resolution of the mutant structure allows for a more definitive placement of the carbonyl of Pro11. The consequence of this change is that there is no torsion angle strain at residue 16. This result suggests that there is no active site torsion angle strain in wild-type E. coli HPr. The lack of substantial change at the active center of E. coli HPr Ser46Asp HPr suggests that the effect of the Ser46 phosphorylation in HPrs from Gram-positive bacteria is due to an electrostatic interference with enzyme I binding.